Allergic diseases such as asthma are some of the most common chronic illnesses affecting children in the U.S. and such diseases are increasing in prevalence worldwide. Based on data from the Centers for Disease Control and Prevention, 7.1 million children are currently affected by asthma (American Lung Association 2012)9 and many others suffer from other atopic allergic diseases. New therapies are urgently needed that target TH2 cytokine producing T cells while leaving other arms of the immune system intact. This proposal focuses on Ndfip1, a protein that we discovered limits TH2 cell differentiation and allergic inflammation, and the E3 ubiquitin ligase Itch. Ndfip1 limits TH2 differentiation, in part by promoting Itch ubiquitylation of JunB. However, it is clear that Ndfip1 functions well beyond this interaction. Our new data begins to explore how Ndfip1 works. These data reveal novel modes of ubiquitin pathway activation that suggest therapeutic strategies that could be useful in treating allergic diseases such as asthma. Our preliminary data defines a new paradigm for the activation of Itch and other closely related family members. In this proposal, we will answer important unresolved questions explaining how Ndfip1 activates Itch and related E3s (Aim 1). These studies will define the function of individual WW domains in relief of Itch autoinhibition, Ndfip1 activation, and interaction with substrates, thus making allosteric therapeutic design possible. In Aim 2 we will identify and analyze substrates of Ndfip1/Itch ubiquitylation. Identifyig substrates of ubiquitin pathways has been enigmatic. However, tools have recently been developed that now make this possible (see Aim 2). Here we will use two newly available enrichment strategies to identify substrates while also determining the ubiquitylated lysine residue(s). We have tested both techniques and have already identified several promising new substrates including TCF1 and PKCa. In Aim 3, we will test these and other newly identified substrates for their roles in T cell proliferation, TH2 differentiation and IL-4 production assays n vitro and in vivo. Additionally, we will use mouse models of asthma to determine the feasibility of therapeutic targeting of this pathway to treat asthma. Thus, these studies will have a significant and long-term impact as they define the mechanistic basis of how ubiquitin complexes are activated and how they regulate allergic disease. Such information will result in new technological advances while defining allosteric interactions for rational therapeutic design